Localization of dimer formation-negative and pathogenic mutants of DJ-1.

<p>A. DJ-1(−/−) cells were transfected with HA-tagged wild-type, C53A and C106S DJ-1. At 25 hrs after transfection, mitochondria in cells were stained with MitoTracker Red. The cells were then fixed with 4% paraformaldehyde and with 0.2% triton X-100 and reacted with an anti-HA antibody. After cells had been reacted with an FITC-conjugated secondary antibody, proteins were visualized by using a confocal laser microscope as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054087#s2" target="_blank">Materials and methods</a>. B. DJ-1(−/−) cells that had been transfected as described in the legends for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054087#pone-0054087-g003" target="_blank">Figure 3A</a> were fractionated by using a subcellular fractionation kit. Proteins in each fraction were analyzed by Western blotting as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054087#pone-0054087-g002" target="_blank">Figure 2C</a>. C and D. DJ-1(−/−) cells (C) and HeLa cells (D) were transfected with FLAG-tagged wild-type, M26I and L166P DJ-1, and localization of proteins was examined as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054087#pone-0054087-g003" target="_blank">Figure 3A</a>.